Enzymes are proteins that are capable of catalyzing chemical transformations. Enzymes position a substrate or substrates in an optimal configuration and stabilize the transition state in the reaction pathway, thereby determining which of several potential chemical transformations actually occurs. Enzymes can be highly specific, both in terms of the reaction that occurs and in their choice of substrate. Enzymes often accelerate reactions by factors of more than a million. Because of their specificity and catalytic power, enzymes are increasingly being used for industrial applications.
One family of enzymes that is especially useful for industrial applications is the family of oxidoreductase enzymes. Oxidoreductases catalyze redox reactions, such as the reduction of aldehydes and ketones to alcohols, the reductive amination of ketones, aldehydes, and ketoacids to amines and amino acids, the reduction of disulfides to thiols, the reduction of alkenes to alkanes and the like. These reactions are normally reversible, and frequently the same enzymes catalyze the corresponding oxidation reactions. For example, alcohol dehydrogenases and carbonyl reductases catalyze both the reduction of aldehydes and ketones to alcohols and the oxidation of alcohols to aldehydes and ketones. Amino acid dehydrogenases catalyze the oxidation of amino acids to 2-ketoacids and the reductive amination of 2-ketoacids in the presence of ammonium salts to amino acids. Similarly, disulfide reductases catalyze the oxidation of thiols to disulfides or mixed disulfides. Reduction and oxidation reactions are collectively referred to herein as “redox reactions.”
Some oxidoreductases can be used to produce fine and specialty chemicals, and are especially useful for producing chiral intermediates in the pharmaceutical and agricultural industries. Oxidoreductases, like many other enzymes, require other molecules, such as cofactors and cosubstrates, for optimal activity. For example, mixed function oxidases use nicotinamide cofactors as part of the complex catalysis of a hydroxylation reaction for the production of chiral alcohols.
Although a number of different enzymes are known, the development of new applications for enzymes such as oxidoreductases requires an expanded search for new enzymes that catalyze specific reactions of interest. For example, amino acid dehydrogenases that reductively aminate certain 2-ketoacids to naturally occurring L-amino acids are known, but no suitable amino acid dehydrogenase has been identified for the production of many non-naturally occurring amino acids. The enzyme catalyzed reductive amination of ketones that are not 2-ketoacids is comparatively quite rare. Similarly, the stereoselective reduction of ketones catalyzed by alcohol dehydrogenases, ketoreductases and carbonyl reductases is known for certain ketones, but enzymes are not available for catalyzing this reaction with many desired target ketones. Transaminases are known that catalyze the transamination of many 2-ketoacids to alpha-amino acids, but certain target 2-ketoacids, particularly those corresponding to non-naturally occurring amino acids, are transaminated poorly, if at all.
There are several known methods to generate potential enzymes that catalyze specific reactions of interest. For example, diverse populations of enzymes can be found in microorganisms harvested from different environments. These microorganisms can be cultured, and their DNA extracted, amplified by PCR, and cloned into a host for expression of the enzymes. Alternatively, various molecular biology techniques, such as mutagenesis, shuffling, molecular breeding, and gene reassembly, can be used to create vast numbers of mutant versions of an enzyme encoded by a known gene. Examples of gene shuffling and molecular breeding are described in U.S. Pat. No. 5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No. 5,830,721; U.S. Pat. No. 5,837,458; U.S. Pat. No. 5,965,408; U.S. Pat. No. 5,958,672; U.S. Pat. No. 6,001,574; and U.S. Pat. No. 6,117,679, all incorporated herein by reference. Examples of methods for constructing large numbers of mutants are described in U.S. Pat. No. 6,001,574; U.S. Pat. No. 6,030,779; and U.S. Pat. No. 6,054,267, also incorporated herein by reference.
Once potential enzymes that may be able to catalyze specific reactions of interest have been generated, the enzymes are tested for activity on the desired substrate, or target compound. Because many enzymes such as oxidoreductases require nicotinamide cofactors for optimal activity, detection of the oxidation or reduction of the cofactor can be used as a signal of enzyme activity.
Currently, the most common method of detecting enzymes using nicotinamide cofactors involves the direct measurement of the cofactor. For example, as a carbonyl reductase reduces a carbonyl group, the concomitant oxidation of reduced nicotinamide, i.e., the conversion of a reduced form of nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide (NAD+) or the conversion of a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP+) can be detected using the absorbance of the reduced form of the cofactor. This reaction of the cofactor can be monitored in a solution by observing the decrease in the absorbance of the solution at 340 nm using a spectrophotometer. Alternatively, during the carbonyl reductase-catalyzed oxidation of an alcohol to the corresponding aldehyde or ketone, NAD+ is converted to NADH or NADP+ is converted to NADPH. The reduction of the cofactor can be detected by monitoring the increase in absorbance at 340 nm, corresponding to the increase in concentration of reduced nicotinamide cofactor. Similarly, fluorescence measurements of nicotinamide cofactors can be performed as well. Additionally, the change in concentration of oxidized or reduced nicotinamide cofactor can be used to detect other enzymes catalyzing cofactor-requiring reactions of interest.
Detection of enzymatic activity is often performed on many enzyme sources for a particular reaction of interest in a process called screening. Often when screening, and particularly when carrying out high throughput screening, mixtures of cells or cell lysates containing suspended insoluble material are used as potential sources for new enzymes because clarification of the crude mixtures is operationally difficult. The difficulty in using such crude mixtures for routine screening for nicotinamide cofactor-using enzymes is that the reaction mixtures contain suspended solid material in the form of cells or cell debris. This insoluble material impedes the transmission of light through the solution and causes high background readings in the absorbance measurements of the cofactors. The crude mixtures also contain various cellular metabolites and biochemicals that absorb at 340 nm, further compromising the accuracy of the measurements. These issues are even more problematic when using high throughput screening methods due to the small volumes used in high density array formats such as microtiter plates or chips. Similarly, if fluorescence measurements are carried out, detecting the emission of fluorescence is also impeded by the presence of insoluble material.
As an alternative, the products of the desired enzymatic reaction can be detected directly by chromatographic techniques. This method requires sampling each individual reaction followed by chromatographic separation of the reaction products, which may include alcohols, carbonyl compounds, and the like. Such a procedure is complex and time-consuming and is impractical for high throughput screening assays when many enzyme sources are tested for the desired enzymatic activity.